U.S. patent application number 11/096311 was filed with the patent office on 2005-10-06 for frequency comb analysis.
This patent application is currently assigned to Menlo Biocombs, Inc.. Invention is credited to Blume, Frederick R., Haensch, Theodor W., Holzwarth, Ronald, Mei, Michael.
Application Number | 20050219540 11/096311 |
Document ID | / |
Family ID | 23087485 |
Filed Date | 2005-10-06 |
United States Patent
Application |
20050219540 |
Kind Code |
A1 |
Haensch, Theodor W. ; et
al. |
October 6, 2005 |
Frequency comb analysis
Abstract
Methods and apparatus for generating a frequency comb and for
its use in analyzing materials and in telecommunications. The
frequency comb is generated by passing pulsed light from a laser
through an optical fiber having a constriction. The frequency comb
comprises a plurality of monochromatic components separated in
frequency by a substantially constant frequency increment. The
monochromatic components are used to probe materials for analysis.
In preferred embodiments, the materials are DNA, RNA, PNA and other
biologically important molecules and polymers. Optical responses
are observed and used to analyze or identify samples. In
telecommunication applications, the individual monochromatic
components serve as carriers for individual communication channels
that can carry information of any of a variety of types, such as
voice, data and images.
Inventors: |
Haensch, Theodor W.;
(Munich, DE) ; Mei, Michael; (Munich, DE) ;
Holzwarth, Ronald; (Munich, DE) ; Blume, Frederick
R.; (Wenham, MA) |
Correspondence
Address: |
EDWARDS & ANGELL, LLP
P.O. BOX 55874
BOSTON
MA
02205
US
|
Assignee: |
Menlo Biocombs, Inc.
|
Family ID: |
23087485 |
Appl. No.: |
11/096311 |
Filed: |
March 31, 2005 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
11096311 |
Mar 31, 2005 |
|
|
|
10121983 |
Apr 12, 2002 |
|
|
|
6897959 |
|
|
|
|
60283773 |
Apr 13, 2001 |
|
|
|
Current U.S.
Class: |
356/432 |
Current CPC
Class: |
G01J 3/10 20130101; G01N
21/255 20130101; H04B 10/503 20130101; G01N 21/31 20130101; H04B
10/506 20130101 |
Class at
Publication: |
356/432 |
International
Class: |
G01N 021/00 |
Claims
What is claimed is:
1-22. (canceled)
23. A method for transmitting information, the method comprising
the steps of: generating a frequency comb comprising a plurality of
monochromatic spectral lines; encoding information using at least
one of said spectral lines as a carrier; and transmitting said
information to a receiver via an optically transmissive medium.
24. (canceled)
25. The method of claim 23, wherein said information is selected
from the group consisting of textual information, graphical
information, tabular information, visual information, and auditory
information.
26-31. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims the benefit of U.S. provisional
application Ser. No. 60/283,773, filed Apr. 13, 2001, which
application is incorporated by reference herein.
FIELD OF THE INVENTION
[0002] The invention relates to methods and materials for analyzing
and characterizing samples and for improving optical
communications.
BACKGROUND OF THE INVENTION
[0003] Optical frequency pulsing has a host of diverse uses.
However, there are two principal categories into which optical
frequency pulsing may conveniently be placed. A first has to do
with using frequency pulsing to encode information for transmission
across fiber optic lines. A second broad category of use for
optical frequency pulsing is the identification of physical
properties of molecules. Each of these uses of optical frequency
pulsing has limitations that have prevented full exploitation of
the technology.
[0004] For example, a limitation in the use of optical frequency
pulsing for transmission of information is the relatively wide
bandwidth of individual frequency pulses, resulting in overlapping
pulses over large transmission distances. As frequency pulses
proceed along a fiberoptic line, pulse width increases. Over fairly
modest distances, the overlap of frequency lines can result in a
loss of digital information content. Another limitation is the
difficulty in generating a plurality of different, closely spaced
frequencies which limits signal resolution.
[0005] The identification and characterization of physical
substrates using frequency pulses is limited by the ability to
provide sufficiently narrow, stable pulses at high frequency in
order to obtain precise physical chemical resolution of the target
substrate. Typical methods for optical analysis of substrates
involve interferometric measurements. Such measurements necessarily
result in decreased resolution in space and time. Therefore,
interferometric measurements are less than ideal.
[0006] There is a need in the art to provide optical frequency
pulse methodology that provides pulses having narrow line width,
wide spectral width and high repetition rate in order to enable
efficient optical communications and detection high-resolution
detection of physical substrates.
SUMMARY OF THE INVENTION
[0007] The present invention provides frequency combs, and
materials and methods for generating frequency combs and methods of
their use. The invention is useful for both optical analysis of
physical substrates and optical communications. Methods of the
invention comprise use of optical frequency combs to generate a
series of discrete emission lines that can extend across the
optical spectrum or a portion thereof. Because the frequency lines
are narrow, are spaced-apart, and cover a suitable frequency
bandwidth, they are useful to conduct optical communications over
long distances as well as to generate precise light frequencies
used in substrate identification and analysis.
[0008] According to the invention, a frequency comb is generated by
firing a laser pulse into a photonic structure having a small
cross-section, preferably a tapered photonic tube or fiber,
resulting in a series of spaced apart, preferably regularly
spaced-apart, discrete emission lines extending across at least a
portion of the spectrum produced by the original laser pulse. The
frequency comb represents the Fourier transform of a train of short
pulses of a single wavelength of light. In a preferred embodiment,
the frequency comb is generated using a train of laser pulses, each
pulse having a duration of between 10.sup.-12 and 10.sup.-18
seconds, preferably between 1 picosecond and 1 femtosecond, that is
passed through an optical fiber having a constriction between about
1.8 microns to about 1.5 microns, and preferably about 1.7 microns
diameter.
[0009] In one embodiment, at least one monochromatic component or
spectral line of the frequency comb, once generated, is focused by,
for example a lens, onto a substrate for identification and/or
analysis. The frequency comb may be focused to a single point for
detection of a discrete molecular entity. For example, in a
preferred embodiment, frequency combs are used to determine the
identity of individual nucleic acids in a linearized strand of
deoxyribonucleic acid (DNA). The use of frequency combs enables
detection of the sequence of DNA where detection with single-source
light would not be possible. Each monochromatic component of the
frequency comb comprises photons having energy E that is
proportional to frequency, represented by E=h.nu.. A substance to
be analyzed, such as a nucleic acid, absorbs a photon of a specific
frequency corresponding to an energy transition in the nucleic
acid. Observation of absorption of photons of the specific
frequency provides information about the identity of the nucleic
acid in question. Pulses of extremely short duration, such as
pulses in the range of 1 picosecond to less than 10 femtoseconds,
interact with the substance in time periods so short that the
substance has insufficient time to undergo chemical reaction or
even to vibrate. Accordingly, frequency combs of the invention
allow a selection of frequency components best suited to identify
the target sample. In fact, the number of emission lines produced
by the average frequency comb, along with the ability to rapidly
pulse a sample, allows redundant probing of the sample to increase
the specificity of detection. A frequency spacing of approximately
10 GHz between monochromatic components of a frequency comb
generated with a laser having a wavelength of approximately 800 nm
has monochromatic components that differ in wavelength by
dimensions of the order of 0.1 Angstroms.
[0010] A sample may be exposed to multiple illumination prior to
detection. Illumination of a sample preferably comprises pulsed
illumination at a frequency in the range of approximately 10.sup.12
per second to approximately 10.sup.18 per second. The pulsed
illumination preferably has a pulse duration that is in the range
of about one picosecond to less than about ten femtoseconds. In a
more preferred embodiment, the pulse duration is less than about
one femtosecond. In an alternative embodiment, the illumination is
continuous.
[0011] Due to the nature of the invention, the invention may be
used to detect any sample upon or through which light can be
projected. Similarly, the invention can be used to elucidate any
sample characteristic, including but not limited to the identity of
the sample, its chemical composition, its state, its
three-dimensional structure, its phase, and similar
characteristics.
[0012] Methods of the invention generally comprise an illuminating
step. Illuminating can encompass many forms. For example, the
illuminating step may comprise illuminating the sample with a
plurality of spectral lines in sequence. Alternatively, the
illuminating step comprises the step of combining a plurality of
spectral lines.
[0013] The sample may be prepared for analysis prior to exposure to
the frequency comb.
[0014] For example, samples may be prepared by treatment with a
chromophore. Alternatively, the sample may be labeled with labels
other than a chromophore that can interact with light, for example
a substance having optical rotatory power. The sample for analysis
can be selected from a variety of substances, including tissue,
nucleic acid, protein, cells, metal, minerals, and other molecules
and compositions. The determining step of the analysis includes
comparing the response to one or more responses, each associated
with a known characteristic, which can be determined empirically or
by reference to known standards.
[0015] In another aspect, the invention features a method for
determining the identity of a sample. The method comprises the
steps of generating a frequency comb comprising a plurality of
monochromatic spectral lines, illuminating a sample with at least
one of the spectral lines, detecting a response to the illuminating
step produced by the sample, and determining the identity of the
sample based upon the response. In methods of determining the
identity of a sample, the determining step comprises comparing the
response to one or more responses, each known to be associated with
an identified sample.
[0016] In either the method of analyzing a sample or the method of
determining the identity of a sample, the step of generating a
frequency comb can involve passing monochromatic light through an
optical waveguide having at least one constriction therein.
[0017] A preferred method of using frequency combs of the invention
is the sequencing of a polymer, such as DNA. In general, a
linearized DNA is passed through a channel comprising a detection
zone. Light (i.e., at least one of the multiple emission lines)
produced by the frequency comb is concentrated at the detection
zone where it comes into contact with a single nucleotide.
Preferably, the single nucleotide is pulsed by the frequency comb
at a desired frequency. At least one spectral response produced
from the nucleotide is detected by a spectrometer, a
spectrophotometer, a CCD detector, or similar device for the
detection of light spectra. The detected spectral response is
characteristic of the nucleotide that was probed by the frequency
comb. By repeating this procedure over the length of a
single-stranded DNA, the sequence of the DNA is determined with
high accuracy.
[0018] More specifically, the DNA to be analyzed is passed through
a transport channel for analysis. The channel is designed to allow
DNA to pass through in a substantially uncoiled, or linear,
conformation so that each or substantially all the individual
nucleotides of the DNA sweep past a location at which interaction
occurs between the DNA and one or more discrete monochromatic
components of a frequency comb. The results of the analysis provide
sequential identification of individual nucleic acids. The DNA can
be held in a substantially linear shape by the use of channels in
any of a variety of materials, as is described in the literature
referred to in more detail below. The skilled artisan appreciates
that methods of the invention are useful to analyze any polymer and
to obtain the sequence of the monomer units. Examples of other
polymers include proteins, nucleic acids, sugars, synthetic
polymers and others.
[0019] The invention also contemplates the detection of molecular
structure in single molecules, in groupings of biological interest,
such as codons and genes or specific information bundles by the
methods and systems disclosed. The detection process uses
recognition patterns obtained from known molecules and sequencing
the information generated for a record or data base.
[0020] Systems and methods of the invention provide detectors that
read, measure, quantify, and detect alterations in various parts or
the continuum of a frequency comb used to identify, analyze or
quantify molecules, nucleotides, binding sites, abnormalities,
sizes and dimensions of targeted biological or molecular
structures. In one embodiment, the detector detects missing
segments of the light spectrum that are removed either by
scattering processes or by absorption.
[0021] The detected spectral response may be any light spectrum.
Preferred spectra include absorption, reflection, transmission,
fluorescent, and chemiluminescent spectra produced through
interaction of the frequency comb, or portions thereof, with the
target. The spectrum produced by a detection target may be compared
to a standard in order to identify the target or its physical or
chemical properties.
[0022] Due to the speed with which light travels, and the extremely
short duration of pulses used, methods of the invention are useful
to analyze samples in any state of matter. Thus, gases, liquids,
solids, and transitional states are measurable and detectable using
methods of the invention. Preferred samples for analysis using
methods of the invention include organic and non-organic
substances, biological compounds, molecules, atomic structures, and
any other compositions upon which light can be projected. In a
preferred embodiment, methods of the invention are used to analyze
a biological molecule, such as a nucleic acid, including DNA,
ribonucleic acid (RNA), peptide nucleic acid (PNA), proteins, or
analogs of nucleic acids or proteins. For flow velocities of
liquids or gases of conventional value, such as rates in the range
of tenths of millimeters per second to meters per second (i.e., a
flow rate spanning four orders of magnitude), light pulses
operating at rates of 100 MHz (i.e., 10.sup.8 per second) would
sample a flowing substance at a differential position or spacing of
from 0.01 Angstrom to 10 Angstroms, or 10.sup.-3 nm to 1 nm.
[0023] Methods and materials of the invention are useful to
catalyze and to monitor chemical reactions. Light pulsing using
frequency combs of the invention is useful to "visualize", through
the appropriate spectrum produced by interaction of the elements of
a chemical reaction with the frequency comb, the process of a
chemical reaction and its components. In one embodiment, multiple
frequency combs are used to probe a chemical reaction along the
length of a reaction tube or chamber in order to analyze different
portions of the reaction.
[0024] One of ordinary skill in the art appreciates that there are
many alternative configurations that can be used for analyzing
physical substrates, both with regard to the substrates and
materials to be analyzed, and with regard to the methods and
apparatus used to perform the analysis. The invention contemplates
any analytical method that relies on the use and detection of
optical signals from the ultraviolet through the visible and
extending to the infrared. The invention contemplates any feature
of optical technology that can provide meaningful information about
a substrate or material, including but not limited to absorption,
transmission, reflection, refraction and emission of light,
observation of features of light such as polarization, phase, index
of refraction, transmission velocity, interference, and energy
transfer by optical methods.
[0025] Methods and materials of the invention are also useful in
optical communication technology. In one embodiment, the
communication technology is a conventional fiber optic based
telecommunication methodology. Each of the large number of
frequencies of light present in frequency combs of the invention is
useful as a carrier for a separate communication channel, in a
system such as a wavelength division multiplexing (WDM) system. A
single optical fiber of the conventional type used for
telecommunications can support thousands of individual
communications simultaneously, each transmitted on a discrete
channel. In one embodiment, a communication of information, whether
in the form of voice, data, or image, or some combination thereof,
is performed using a monochromatic component of a frequency comb as
a carrier upon which the information is modulated. The modulated
carrier is transmitted by a transmitter and received by a receiver.
The information is demodulated from the carrier by the receiver,
and the information is provided to a recipient. One of ordinary
skill appreciates that the information can be modulated onto the
carrier as digital or analog information, and that the information
can be transmitted or received by a person using the appropriate
output or input device, or by a machine such as a computer or fax
machine.
[0026] In practice, the invention features methods for transmitting
information. Methods of the invention comprise the steps of
generating a frequency comb comprising a plurality of monochromatic
spectral lines, encoding information using at least one of the
spectral lines as a carrier, and transmitting the information to a
receiver via an optically transmissive medium.
[0027] In yet a further aspect, the invention features a method for
receiving optically transmitted information. The method involves
receiving information encoded in at least one monochromatic
spectral line, wherein the line is a spectral component generated
by passing monochromatic illumination through an optical waveguide
having one or more constrictions therein.
[0028] In some embodiments, the method of transmitting information
or the method of receiving information can employ information that
is selected from the group consisting of textual information,
graphical information, tabular information, visual information, and
auditory information.
[0029] Further aspects and features of the invention are presented
in the following drawings and detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0030] The objects and features of the invention can be better
understood with reference to the drawings described below, and the
claims. The drawings are not necessarily to scale, emphasis instead
generally being placed upon illustrating the principles of the
invention. In the drawings, like numerals are used to indicate like
parts throughout the various views.
[0031] FIG. 1 is a schematic diagram of an exemplary apparatus used
to synthesize a frequency comb using a single laser source,
according to principles of the invention;
[0032] FIG. 2 is a close-up view of an exemplary tapered optical
fiber having a constriction therein, according to principles of the
invention;
[0033] FIG. 3A is a schematic diagram showing an exemplary
analytical apparatus, according to principles of the invention;
[0034] FIG. 3B is a diagram showing an exemplary optical cavity
that causes illumination to pass a specimen a plurality of times,
according to principles of the invention;
[0035] FIG. 4 is a diagram showing the frequency comb 40 shown in
FIG. 1 in greater detail; FIG. 5 is an exemplary schematic energy
diagram showing the relationship between potential energy and
interatomic distance in a molecule; and
[0036] FIG. 6 is a diagram showing an exemplary application of a
frequency comb to measuring frequencies of optical sources using a
cesium clock microwave standard.
DETAILED DESCRIPTION OF THE INVENTION
[0037] The invention provides frequency combs for analysis of
materials and for communications. A frequency comb is a plurality
of narrow, spaced-apart light emission lines produced from an
essentially monochromatic light source. Frequency combs are useful
because they provide discrete wavelengths of light separated in
space and time for very accurate transmission of information
(either information concerning a substrate to be analyzed or
optically-encoded information content).
[0038] The present invention provides frequency combs that span the
entire spectrum, preferably in frequency ranges of the order of an
octave having extremely precise wavelengths with very narrow
frequency separations between individual pairs of discrete
monochromatic components. Frequency combs of the present invention
permit the precise measurement of characteristics of materials that
are measurable using absorption, emission, reflection, refraction
and transmission. In particular, frequency combs of the present
invention provide extremely large numbers of discrete lines. In
some embodiments the frequency comb comprises millions of discrete
monochromatic components or lines. These discrete monochromatic
components can be used individually, or they can be combined, to
form optical sources that can be tuned to match the characteristics
of individual chemical moieties such as atoms, molecules, chemical
functional groups, chemical monomers, polymers, ions, salts and/or
adducts of molecules. Such optical sources can be designed to
excite a response from a known material and to elicit no response
or a much diminished response from another known material, thereby
providing a convenient method of discrimination between the two
materials, or alternatively, a convenient method of analyzing a
material for the presence of one or another known chemical
moiety.
[0039] In practice there are two preferred applications of
frequency combs. The first is in the area of substrate analysis.
Because frequency combs comprise narrow emission lines that can be
pulsed onto a substrate at high frequency, they are useful to
detect, identify, and characterize substrates that are not amenable
to elucidation with conventional single wavelength light. For
example, frequency combs are useful to determine the sequence of
nucleic acids, such as DNA, RNA, and PNA. A typical nucleic acid in
DNA is of the order of tens of angstroms wide. A typical wavelength
of light in the visible parts of the spectrum, such as the
wavelength of the maximal intensity of sunlight, has a wavelength
of thousands of Angstroms, or hundreds of nanometers. Thus, no
matter how rapidly such a wavelength is pulsed onto a nucleic acid,
it will never "see" the nucleic acid at a resolution fine enough to
distinguish one nucleotide from another (e.g., an adenine from a
thymine). Frequency combs have discrete monochromatic components
that differ in wavelength, in some embodiments, by only about 0.01
angstroms. The duration of an individual pulse can range from
approximately 10.sup.-12 seconds (picoseconds) to 10.sup.-18
seconds (attoseconds). Thus, frequency combs obtain time resolution
of individual monomer components of a polymer, such as DNA, by
virtue of their size, selection, and pulse rate.
[0040] In some embodiments, femtosecond (10.sup.-15 seconds) pulses
coupled with the narrow bands produced in a frequency comb allows
detection of nucleic acid sequences through the ability to
differentiate the various nucleotides that make up the structure of
the nucleic acid. One useful range of pulse durations is the range
of about 1 picosecond to less than about 10 femtoseconds. In one
embodiment, the differentiation occurs based on the ability or lack
thereof of a nucleic acid to interact with a specific wavelength of
light. An example is absorption of a discrete wavelength associated
with a change in the energy state of the nucleic acid. In another
embodiment, the differentiation occurs based on the brevity of
duration of a pulse, which at the femtosecond time scale is of the
same duration as the time for a molecule to vibrate or to begin to
react chemically.
[0041] Methods of the invention for sequencing nucleic acids
comprise generating a frequency comb as described in detail below
and in the figures. A nucleic acid molecule is then linearized
using methods known in the art. See, e.g., PCT Published Patent
Application WO 96/29593, incorporated by reference herein. The
linearized nucleic acid is passed through a channel having a width
approximately equal to, but larger than, the width of the
linearized DNA. The channel comprises a detection zone in which
each nucleotide sequentially passes as the DNA proceeds through the
channel. At the detection zone, each nucleotide, or a group of
nucleotides, is pulsed with at least one monochromatic component of
a frequency comb as described below. An optical response is then
measured from each nucleotide, or from a group of nucleotides. The
cumulative response based on the pulsed light provides a signature
for each nucleotide, or group of nucleotides, in the sequence.
Details are provided below.
EXEMPLIFICATION
Example 1
Generation of a Frequency Comb
[0042] Referring to FIG. 1, a source 10, such as a Titanium (Ti):
sapphire (Al.sub.2O.sub.3:Ti.sup.3+) laser, operated in pulsed
mode, for example at a frequency of 625 MHz, with a pulse duration
of 25 femtoseconds (fs), provides illumination that is focused by a
lens 20 into an optical path. The optical path can be an optical
fiber 30, such as the tapered optical fiber shown in greater detail
in FIG. 2, that has a constriction 32. In one embodiment, the
tapered optical fiber 30 has an overall diameter of about 125
micrometers and a constriction 32 (or "waist") having a diameter
that is 1.8 micrometers in diameter. In some embodiments, the waist
32 is 1.5 micrometers in diameter. The interaction of the pulsed
illumination with the constriction 32 creates a frequency comb 40
("frequency comb illumination"). The frequency comb illumination
exiting the tapered optical fiber 30 is focussed by a lens 35. In
one embodiment, the frequency comb illumination contains optical
radiation that comprises a series of substantially monochromatic
signals having a frequency spacing of about 10 GHz.
[0043] The pulse train is controlled at a desired frequency by the
clock 38, which controls the pulse rate of the mode-locked laser.
The pulses from a mode-locked laser are produced in a periodic
train. Therefore, the broad frequency spectrum of the laser is
composed of a vast array, or comb, of distinct frequency modes
spaced by the cavity repetition rate. In principle, a single pulse
would contain an infinite number of distinct frequency modes. The
repetition rate R is equivalent to the frequency-domain comb
spacing of the emitted pulse train. The repetition rate is
determined by the cavity length, L, and the group velocity,
v.sub.g, of the intracavity pulse according to the relation
R=v.sub.g/(2L), or velocity divided by round trip distance. In an
exemplary embodiment, a repetition rate is selected by controlling
the cavity length, and hence the round trip distance, for example
with a transducer such as a piezoelectric transducer that controls
a cavity mirror and a control loop that senses a selected harmonic
of the repetition rate so as to obtain phase lock.
[0044] In one embodiment, a selected substantially monochromatic
optical signal impinges on a second harmonic generating crystal 50,
such as a 7 millimeter segment of Potassium Titanyl Phosphate
(KTP). The frequency doubled radiation emitted therefrom is
combined with a second selected frequency, which provides sum and
difference radiation frequencies ("beats"), the spacing of which is
indicative of the frequency spacing in the frequency comb.
[0045] FIG. 4 is a diagram showing the frequency comb 40 shown in
FIG. 1 in greater detail. The frequency comb 40 comprises a
plurality of discrete wavelengths .nu..sub.1 42, .nu..sub.2 42',
.nu..sub.3 42". The discrete wavelengths .nu..sub.1 42, .nu..sub.2
42', .nu..sub.3 42" are separated one from the other by a spacing
.DELTA..nu. 44 given as a typical example by
.DELTA..nu.=.nu..sub.2-.nu..sub.1. The range of frequencies in the
frequency comb 40 can span an octave (i.e., the highest frequency
is at least twice the lowest frequency), or equivalently, in
wavelength, the shortest wavelength is no longer than one half the
longest wavelength. Frequency combs 40 having more than an octave
of bandwidth are possible.
Example 2
Use of a Frequency Comb in Biologically Important Applications
[0046] As shown schematically in FIG. 3A, the frequency comb
illumination may be focused onto substantially a point of light 65
that coincides with a point within a channel 60 through which a
fluid 70 can flow, as indicated by the arrow 70. The channel is
fabricated from a suitably transparent material. The channel 60
carries a fluid 70 comprising a chemical substance to be identified
or that is intended to otherwise react with at least a portion of
the frequency comb illumination. For example, a solution containing
DNA may move down the channel 60 into the illuminated area. As will
be discussed in more detail below, the DNA can be caused to move
under the action of electric fields, or alternatively the DNA can
be carried by a moving carrier fluid. As the frequency comb
illumination passes through the channel 60, one or more frequency
components of the frequency comb interact preferentially with the
chemical substance, such as the individual nucleic acid bases of
the DNA. In some embodiments, the interaction is a specific
absorption of certain peaks in the frequency comb which are
characteristic of the substance. In other embodiments, the
interaction can be the excitation of a response from the chemical
substance such as when a moiety is labeled with a chromophore. In
addition the response may be chemical, such as a chemical reaction,
or physical, such as an optical reemission at a specific
wavelength. The frequency comb illumination passing through the
channel 60 can be detected or observed, with or without beam
shaping or focusing with a lens 80. The transmitted frequency comb
illumination can for example be observed using a spectrometer or
spectrophotometer 90.
[0047] In some embodiments, a passageway having an effective
diameter of less than approximately 5 nanometers (nm) is provided
to maintain single strand DNA material or other polymers of
interest (e.g., DNA) for analysis in a linearized form. The
literature indicates that linear single-stranded DNA has a diameter
of about 1.6 nm, and linear double-stranded DNA has a diameter of
about 3.4 nm. The literature indicates that dual-strand DNA in its
natural, or folded, configuration is larger that 5 nm in dimension
and so will not pass through a passageway of less than 5 nm
effective diameter. Equivalently, a passageway of less than 5 nm
effective diameter is too narrow to permit the folding of a
linearized single or double strand of DNA, The literature describes
a number of materials that define suitable passageways or apertures
therein. Passages can be generated in flat plate by damaging the
plate material, for example by bombardment with charged particles,
followed by etching. See for example, R. L. Fleischer, P. B. Price,
R. M. Walker, Nuclear Tracks in Solids (Univ. of California Press,
Berkeley, Calif. (1975); European patent Application No.
83305268.1; and U.S. Pat. Nos. 3,303,085; 3,662,178; 3,713,921;
3,802,972; 3,852,134, 4,956,219, 5,462,467, 5,564,959 and
5,449,917, each of which is incorporated herein by reference in its
entirety.
[0048] Various materials comprise passageways or apertures of
suitable size when they are manufactured. Examples include arrays
of carbon nanotubes (see Iijima, Nature, 354:56 (1991); U.S. Pat.
No. 5,457,343; U.S. Pat. No. 5,346,683), and anodic porous alumina
(see A. Despic and V. P. Parkhutik, in Modern Aspects of
Electrochemistry, J. 0. Bockris, R. E. White, B. E. Conway, Eds.
(Plenum, New York, 1989), vol. 20, chap. 6; D. AlMawiawi, N.
Coombs, M. Moskovits, J Appl. Phys. 70, 4421 (1991); Martin, C. R.,
Science, 266:1961 (1994)). In addition, anodic porous alumina has
been used as a template for making metal structures having the same
shape and dimensions (see Matsuda and Fukuda, Science, 268:1466
(1995)). Other porous materials with small pores for use as
templates have been described in Ozin, G., Adv. Mater. 4:612 (1992)
and in Nishizawa et. al., Science 268:700 (1995). Each of the
above-mentioned publications is incorporated herein by reference in
its entirety.
[0049] Linearized DNA molecules are generated in fluids under the
influence of electric fields (see Bustamante, C. 1991. Direct
observation and manipulation of single DNA molecules using
fluorescence microscopy. Annu. Rev. Biophys. Biophys. Chem.
20:415-46; Gurrieri, S. Rizzarelli, E. Beach, D. and Bustamante, C.
1990. Imaging of kinked configurations of DNA molecules undergoing
orthogonal field alternating gel electrophoresis by fluorescence
microscopy. Biochemistry 29:3396-3401; and Matsumoto, S., Morikawa,
K., and Yangida, M. 1981. Light microscopic structure of DNA in
solution studied by the 4', 6-diamidino-2-phenylindole staining
method. J. Mol. Biol. 152:501-516. Each of the above-mentioned
publications is incorporated herein by reference in its
entirety.
[0050] Reports of linearized DNA passing through passageways under
the influence of applied electric fields also appear in the
literature (see Kasianowicz, J. J., Brandin, E., Branton, D.; and
Deamer, D. W. 1996. Characterization of individual polynucleotide
molecules using a membrane channel. Proc. Natl. Acad. Sci. USA.
93:13770-3; and Bezrukov, S. M., Vodyanoy, I., and Parsegian, V. A.
1994. Counting polymers moving through a single ion channel.
Nature. 370:279. Each of the above-mentioned publications is
incorporated herein by reference in its entirety.
[0051] As shown schematically in FIG. 3B, the region surrounding
the channel 60' can be an optical cavity 62, 64, within which the
frequency comb illumination makes multiple passes through the
channel 60', so as to increase an interaction cross-section between
the frequency comb illumination and a substance in the channel 60'.
For ease of understanding, a single ray 66 of the frequency comb
illumination is shown in FIG. 3B.
[0052] In some embodiments, the end of the optical fiber 30
comprises nanoparticles, such as gold nanoparticles, that interact
with the illumination by absorption and re-emission. The presence
of nanoparticles modifies the radiation field behavior of the
optical fiber 30.
[0053] The analysis of the radiation that is transmitted through
the channel provides information as to the identity or chemical
composition and the concentration of substances in the channel. The
signature that results from absorption of selected frequency
components of the frequency comb illumination can provide
information as to the identity and quantity of a particular
substance in the channel. In alternative embodiments, a dual beam
geometry can be used to provide a measurement referenced to a
defined condition, such as a known quantity of a known substance
within fluid in a channel.
[0054] Methods and systems of the invention are applicable to a
broad range of materials and to a broad range of measurements.
Titanium sapphire lasers are tunable with an emission band having a
range of approximately 660 nm to approximately 1100 nm. The
frequency of the titanium sapphire laser can be increased by
factors of integers, such as frequency doubling and frequency
tripling the laser light emission, thereby decreasing the emitted
wavelength by factors of 2 and 3, respectively. Accordingly, the
titanium sapphire laser is useful to generate frequency combs over
a range of frequencies. One can also employ other laser sources to
generate frequency combs. For example, some Nd:glass and Ytterbium
lasers generate pulses in the 10-1000 femtosecond range.
[0055] In the field of chemistry, reactions are controlled by
providing sufficient energy to overcome reaction barriers. Chemical
reactions depend on complex combinations of such features as
thermodynamic stability of reagents and products under specific
conditions of pressure, temperature and composition (i.e.,
solvation, pH, the presence of catalysts, and the like), and
detailed molecular features such as energy states including ground
states and excited states and the associated electronic
wavefunction distributions, internuclear or interatomic distances,
molecular conformations, and vibrational modes. The preceding list
is not intended to be exhaustive, but rather to indicate the range
of features that can have an effect on a given chemical reaction.
The path of a chemical reaction and the end products that are
obtained can be controlled by controlling some or all of the
enumerated features, as well as others. In particular, the use of
light having particular frequencies and polarization properties can
affect the energy states of atoms and molecules. Light of a
properly selected frequency and polarization can be absorbed by a
chemical substance, which thereby gains energy corresponding to
E=h.nu.. As is well understood in the spectroscopic arts, the
change in energy can result in a transition to a different energy
state, and/or can result in a change in the interatomic spacing of
atoms in a molecule.
[0056] FIG. 5 is an exemplary schematic energy diagram 500, in
which individual curves 510, 520 represent the relationship between
potential energy and interatomic distance for a specific electronic
state. The diagram is based on theoretical calculations. At a
location 530 in the diagram where two curves 510, 520 come close
together or actually intersect, the molecule can undergo a
spontaneous transition from one energy state to another.
Transitions between curves can also be driven by the absorption or
emission of a photon having the appropriate energy. The diagram
gives as an example data for the material sodium iodide, Nal. In
this example, curve 510 is the ground state and curve 520 is the
first excited state. The point 530 corresponds to an internuclear
distance of 6.9 Angstroms, which represents the internuclear
distance at which a state transition is most likely. For more
complex materials, it may not be convenient or possible to
construct the appropriate theoretical energy diagram. Nevertheless,
the conceptual basis for initiating and driving chemical reactions
is understandable in terms of applying the correct quantity of
energy to a substance in a selected energy state. Prior to the
development of an optical source such as the frequency comb 40,
which provides discrete monochromatic lines having very closely
spaced discrete energies, there has been no practical way to
provide precisely tuned, precisely timed energy pulses suitable for
use in driving a specific chemical reaction involving a particular
molecular entity in a selected energy state.
[0057] The invention contemplates the use of the entire light
spectra (from IR to UV and the entire range of colors in light
combs) to gain molecular and biological information. In one aspect,
methods and systems of the invention provide the ability to detect
biological and molecular information through the use of "light
combs" utilizing the discrete segments in terms of time, distance,
light properties and the entire wavelength of a light comb. In one
embodiment, methods and systems of the invention use light combs to
distinguish biological or molecular information through measured
absorption of specific spectra of the light comb. In one
embodiment, methods and systems of the invention use light combs to
distinguish biological or molecular information through measured,
rapidly activated and redundantly activated chemoluminescence. In
one embodiment, methods and systems of the invention use light
combs to distinguish biological or molecular information through
measured refraction of continuous light wavelength segments. In one
embodiment, methods and systems of the invention use light combs to
distinguish molecular and biological information through rapidly
pulsed discrete fractions of light wavelengths utilizing light
combs. In one embodiment, methods and systems of the invention use
multiple color markers activated by segments of a light comb as a
means of detecting genetic sequences, single nucleotide
polymorphisms, single nucleotides, continuum of nucleotides in a
specific sequence (such as genes) or groups of 3 nucleotides (such
as codons) in DNA or RNA. In one embodiment, methods and systems of
the invention use shaped pulses to excite molecules as a means of
detection of or identification of the molecule based upon a unique
signature of the molecular reaction to its excited state. In one
embodiment, methods and systems of the invention identify molecules
and information about the molecule using selected combinations of
specific color bands from the light comb to elicit a detectable
reaction.
Example 3
Use of a Frequency Comb in Optical Communication Applications
[0058] In the field of telecommunication, an exemplary frequency
comb having a spacing of approximately 50 MHz and a one octave
spectral bandwidth from approximately 600 nm to approximately 1200
nm , which is equivalent to a bandwidth extending from
500.times.10.sup.12 per second or 500 TeraHertz (THz) to 250 THz,
(i.e., 250 THz bandwidth), there would be approximately
5.times.10.sup.6 or 5 million discrete monochromatic components in
the frequency comb. At a frequency separation of 100 MHz, which is
a common separation between adjacent channels in present-day
telecommunication, the frequency comb would support approximately
2.5 million separate channels. A discussion of some of the features
of optical communications appears in U.S. Pat. No. 5,631,758, the
entire disclosure of which is incorporated herein by reference.
Because the repetition rate can be controlled, as described above,
the frequency spacing in the frequency comb can be controlled. For
a comb of a given spectral width, such as an octave, one can
control the number of monochromatic spectral lines by determining a
repetition rate that divides the spectral width into the desired
number of segments. For example, repetition rates can be chosen to
divide the spectral width into, for example, any of 1,000, 10,000,
100,000, 1,000,000 or 10,000,000 segments The number of
monochromatic spectral lines will then be one larger than the
number of segments. As those of skill in the art will recognize,
the only limitation on the repetition rate is that a mode locked
operating condition must be achieved.
[0059] One exemplary use of frequency combs of the invention for
telecommunication employs an individual monochromatic component of
the comb as a carrier upon which information is modulated, and
transmitted using an optical transmission medium such as an optical
fiber, air, or water, depending on the frequency of the
monochromatic component and the optical characteristics of the
transmission medium. A receiver receives the optical communication
and recovers the information by demodulation. The information is
any type of information that can be encoded and transmitted in
analog or in digital form. As is understood by those of skill in
the optical communication arts, general purpose computer hardware
and associated software, or dedicated, custom computer hardware can
be employed to modulate, demodulate, and control the transmission
of information. The information can be transmitted and/or received
using conventional or proprietary data transmission protocols.
[0060] The methods and materials of the invention, including
apparatus that is used to perform the analysis, or to communicate
using frequency combs, are in one embodiment controlled and
operated under computer control, using general purpose computers. A
general purpose computer, is a commercially available personal
computer that comprises a CPU, one or more memories, one or more
storage media, one or more output devices, and one or more input
devices. The computer is programmed with software comprising
commands that when operating direct the computer in the performance
of the methods of the invention. Those of skill in the programming
arts will recognize that some or all of the commands can be
provided in the form of software, in the form of programmable
hardware such as flash memory or ROM, in the form of hard-wired
circuitry, or in some combination of two or more of software,
programmed hardware, or hard-wired circuitry. Commands that control
the operation of a computer are often grouped into units that
perform a particular action, such as receiving information,
processing information or data, and providing information to a
user. Such a unit can comprise any number of instructions, from a
single command, such as a single machine language instruction, to a
plurality of commands, such as a plurality of lines of code written
in a higher level programming language such as C++. Such units of
commands will be referred to generally as modules, whether the
commands comprise software, programmed hardware or hard-wired
circuitry, or a combination thereof.
[0061] In alternative embodiments, the computer is a laptop
computer, a minicomputer, a mainframe computer, an embedded
computer, or a handheld computer. The memory is any conventional
memory such as, but not limited to, semiconductor memory, optical
memory, or magnetic memory. The storage medium is any conventional
machine-readable storage medium such as, but not limited to, floppy
disk, hard disk, CD-ROM, and/or magnetic tape. The output device is
any conventional display such as, but not limited to, a video
monitor, a printer, a speaker, and/or an alphanumeric display
device. The input device is any conventional input device such as,
but not limited to, a keyboard, a mouse, a touch screen, a
microphone, and/or a remote control. The computer can be a
stand-alone computer or interconnected with at least one other
computer by way of a network.
Example 4
Use of a Frequency Comb in Measurement of Optical Frequencies
[0062] FIG. 6 is a diagram 600 showing an exemplary application of
a frequency comb to measuring frequencies of optical sources using
a cesium clock microwave standard. In this example, the frequency
comb generator is augmented with additional hardware to provide an
external reference signal from a cesium clock, as well as laser
illumination whose frequency is to be measured. The frequency comb
generator is similar to that described in FIGS. 1-4 above. In this
example, the generator is the portion of the diagram indicated by
the dotted outline 610. A second harmonic generator appears within
dotted line 620. The remaining portion of the diagram includes the
external standard within dotted line 630, the lasers whose
frequencies are to be measured within dotted line 640, and the
measuring instrumentation within dotted line 650.
[0063] The frequency comb generator comprises a femtosecond laser
612, such as the Titanium-sapphire laser described above, an
optical fiber 614 having a constriction of about 1.7 microns
diameter and a length in the range of 5 to 10 centimeters (cm), a
mode locking device such as a PZT cavity length adjuster 616, and a
pulse rate controller such as the 10 GHz synthesizer 618. The
second harmonic generator 620 comprises a laser sources 622 such as
a Nd: YAG laser and a second harmonic generator crystal 624 such as
KTP. As is understood by those of skill in the optical frequency
arts, the laser 622 emits light at frequency f and the second
harmonic generator absorbs some of the frequency f light and
re-emits light at frequency 2f. The external standard comprises a
reference clock 632 such as the NIST Cesium clocks at Boulder,
Colorado, and a local reference 634, such as a Rubidium clock,
situated near the frequency comb generator. The lasers 642 whose
frequencies are to be measured generate light with frequency
f.sub.a The frequency f.sub.a is what is to be measured. The light
from the laser 642 is added to the optical beam 651 that comprises
the frequency comb and the first and second harmonics f and 2f, The
measuring instrumentation comprises a grating 652 to disperse the
components of the optical beam 651, and two detectors 604 whose
outputs are beat patterns that are combined and are observed by a
counter 656. Not shown is computational equipment that analyzes and
displays various signals to confirm the proper operation of the
apparatus and the test results. The frequency of the laser under
test is determined by computing the offsets of the laser frequency
from known frequencies calibrated with the cesium clock microwave
frequency.
[0064] As is commonly done in the optical arts, components that are
commonly used are shown without, appreciable discussion. These
components include detectors 604 such as photodiodes, mixers 606
denoted by circles containing an "x," and semi-transparent mirrors
608 that denoted by short solid lines placed at approximately 45
degrees to a beam line, appear at several locations in FIG. 6.
Their meaning and use is well known in the art. Arrowheads denote
the direction of propagation of electrical and optical signals in
FIG. 6
Equivalents
[0065] While the invention has been particularly shown and
described with reference to specific preferred embodiments, it
should be understood by those skilled in the art that various
changes in form and detail may be made therein without departing
from the spirit and scope of the invention as defined by the
appended claims.
* * * * *